One potential mechanism by which ethanol might produce its characteristic central nervous system (CNS) depression is through inhibition of excitatory transmission. Glutamate is the most abundant excitatory neurotransmitter present in the CNS. Therefore, we examined the effect of ethanol on glutamate-mediated transmission in primary cultures of cerebellar neurons. Ethanol was a potent and selective inhibitor of the actions of glutamate at the NMDA receptor subtype, and was less effective at the kainate receptor in these cells. To define the site of action of ethanol at the NMDA receptor, interactions of ethanol with several modulators of receptor function were examined. Ethanol-induced inhibition of NMDA-stimulated calcium influx was additive with inhibition produced by phencyclidine (PCP) or magnesium ion, indicating that ethanol does not act at a site within the receptor-gated ion channel. Ethanol also did not alter the EC(50) for NMDA stimulation of calcium uptake, suggesting no direct interaction with the NMDA binding site.However, glycine, which acts at a strychnine-insensitive site to enhance responses to NMDA, and has been suggested to be a co-agonist at the NMDA receptor, was found, at high concentrations, to reverse ethanol-induced inhibition of NMDA-stimulated calcium uptake. Structure-activity studies showed that other amino acids that act as agonists at the glycine site also reversed ethanol inhibition. The data suggest that ethanol may interfere with the concerted actions of glutamate and glycine at the NMDA receptor. The specificity of ethanol's action was investigated by comparing effects of barbiturates and benzodiazepines. Barbiturates were more effective at inhibiting kainate- than NMDA-stimulated calcium influx, while flurazepam had no effect. The differential profile of drug effects at excitatory amino acid receptors may contribute to specific pharmacological actions of these drugs.